Plastid transit peptides

ABSTRACT

The present invention provides novel plastid transit peptides that direct localization of attached moieties (e.g., polypeptides) into plant plastids. The present invention also relates to methods and compositions for localizing polypeptides to plant plastids including, but not limited to, transgenic plant production.

This application is a divisional of U.S. Ser. No. 11/150,054 filed onJun. 9, 2005 (which is now U.S. Pat. No. 7,193,133) which claims thebenefit of U.S. provisional application No. 60/578,535, filed Jun. 9,2004, each of which are incorporated herein by reference in theirentirety.

1. FIELD OF THE INVENTION

The present invention relates generally to the field of proteintargeting and provides peptides that direct localization of attachedpolypeptides into plant plastids. The present invention also relates tomethods and compositions for localizing polypeptides to plant plastidsincluding, but not limited to, transgenic plant production.

2. BACKGROUND OF THE INVENTION

Plastid transit peptides are N-terminal extensions that facilitate thetargeting and translocation of cytosolically synthesized precursorproteins into plastids via a post-translational mechanism (reviewed byBruce, Biochim. Biophys. Acta 1541:2-21 (2001)). With the sequencing ofthe entire Arabidopsis genome now completed, it is estimated that morethan 3500 different proteins are targeted into the plastids during thelife of a typical plant. Developing a model for how all of thesetargeting sequences function to direct proper targeting has beendifficult, since they are highly divergent at the primary sequence levelin terms of length, composition, and organization. Secondary andtertiary structural information is only available for a few plastidtransit peptides, and the results differ significantly depending onwhether the experiments were carried out in an aqueous ormembrane-mimetic environment. Thus, no common structural features orproperties have been clearly delineated.

The capability to target recombinant proteins to different subcellularcompartments in transgenic plants is an important part of plant geneticengineering. For example, many important plant physiological processestake place in plastids including, but not limited to, photosynthesis,fatty acid synthesis, amino acid synthesis, carotenoid biosynthesis,terpenoid biosynthesis, and starch biosynthesis. As such, there is aneed for the ability to target recombinant polypeptides to plastids tomodulate or alter the physiological processes that occur in theplastids. Additionally, some polypeptides are toxic when expressedrecombinantly in the cytoplasm. Because plastids are subcellularcompartments, it is possible to target recombinant polypeptides to theplastids to sequester them from the cytoplasm, thus allowing for higherexpression levels. Furthermore, expression of recombinant polypeptidesin plastids may facilitate isolation of the polypeptide for variousapplications.

3. SUMMARY OF THE INVENTION

The present invention relates to a novel plastid transit peptidesselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57. In addition to thepolypeptide sequences of SEQ ID NOS:1-57, it will be appreciated thatpeptides of the invention also encompass variants thereof, including,but not limited to, any fragment, derivative, or analog thereof.

The present invention also relates to nucleic acid molecules that encodeany one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, and SEQ ID NO:57, or any variants (e.g., any fragment,derivative, or analog) thereof. Nucleic acid molecules that encodepeptides with plastid transit functional activity (e.g., the ability todirect an attached moiety into a plastid) and hybridize under stringentconditions to any of the nucleic acid molecules that encode any of SEQID NOS:1-57 are also encompassed.

Vectors or expression cassettes comprising nucleic acid molecules of theinvention are also encompassed. Cells, plants, or seeds comprising thevectors of the invention are also encompassed.

The present invention also relates to transgenic plants expressing anucleic acid molecule and/or peptide of the invention. The transgenicplants can express the transgene in any way known in the art including,but not limited to, constitutive expression, developmentally regulatedexpression, tissue specific expression, etc. Seeds obtained from atransgenic plant of the invention are also encompassed.

Methods of production of the peptides of the invention and/orpolypeptides comprising one or more peptides of the invention, e.g., byrecombinant means, are also provided. Compositions comprising one ormore peptides of the invention and/or polypeptides comprising one ormore peptides of the invention are also encompassed.

The present invention also provides methods for targeting a polypeptideto a plastid in a plant by attaching a plastid transit peptide of theinvention to the polypeptide to be targeted. In some embodiments, themethod comprises recombinantly attaching a first nucleic acid moleculeencoding a plastid transit peptide of the invention to a second nucleicacid molecule encoding a polypeptide to be targeted such thattranslation of the nucleic acid molecule produces a fusion polypeptide.

Methods of identifying novel plastid transit peptides are encompassed bythe present invention comprising i) introducing into a plant or plantcell a vector comprising a first nucleic acid molecule encoding acandidate plastid transit peptide linked to a second nucleic acidmolecule encoding a polypeptide that is only active in a plastid suchthat translation of the first and second nucleic acid molecule producesa fusion protein and ii) screening for activity of the polypeptide,wherein said activity indicates that the polypeptide is localized to aplastid and the candidate plastid transit peptide is functional.

3.1 Definitions

A “plastid transit peptide” refers to an amino acid sequence thatmediates targeting or localization of an amino acid sequence to which itis attached (e.g., as a fusion polypeptide) to a plastid.

A “plastid” refers to a small, double-membraned organelle of plant cellsand certain protists that contains ribosomes, DNA, and, often, pigment.Plastids can occur in an undifferentiated form (proplastid) and severaldifferentiated forms including, but not limited to chloroplasts,etioplasts, amyloplasts, chromoplasts, elaioplasts, and leucoplasts.

The terms “nucleic acid molecule” or “polynucleotide” refer todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acid molecules containing known analogues ofnaturally occurring nucleotides that have similar binding properties asthe reference nucleotides and are metabolized in a manner similar tonaturally occurring nucleotides.

The terms “polypeptide,” “peptide” and “protein” refer to a polymer ofamino acid residues. The terms apply to amino acid polymers containingnaturally occurring amino acid residues as well as amino acid polymersin which one or more amino acid residues is an artificial chemicalmimetic of a corresponding naturally occurring amino acid (e.g.,non-classical amino acid). The amino acid residues of amino acidpolymers are generally linked by covalent peptide bonds but may belinked by any other method known in the art. As used herein, the termsencompass amino acid polymers of any length, including full-lengthproteins.

The term “amino acid” refers to naturally occurring amino acids,synthetic amino acids, as well as amino acid analogs and mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code.Amino acid analogs include, but are not limited to naturally occurringamino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acids may be referred toherein by either the commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

The term “promoter” refers to regions or sequence located upstreamand/or downstream from the start of transcription that are involved inrecognition and binding of RNA polymerase and other proteins to initiatetranscription. Promoters include necessary nucleic acid sequences nearthe start site of transcription, such as, in the case of a polymerase IItype promoter, a TATA element. A promoter also optionally includesdistal enhancer or repressor elements, which can be located as much asseveral thousand base pairs from the start site of transcription. A“constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

A “vector” refers to a nucleic acid molecule capable of replication in ahost cell independently of and/or integrated into the host chromosome.Vectors may be, e.g., plasmids and may have an origin of replicationand/or expression elements such as transcription/translation initiatorsand terminators and promoters useful for regulation of the expression ofthe particular nucleic acid molecule.

An “expression cassette” refers to a nucleic acid molecule which, whenintroduced into a host cell, results in transcription of a RNAtranscript corresponding to at least a portion of the expressioncassette and translation of a peptide or polypeptide from the RNAtranscript. The nucleic acid molecule may contain a transcriptionalstart and/or stop codon.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like) and cells (e.g. guard cells, egg cells, trichomesand the like), and progeny of same. The class of plants that can be usedin the method of the invention is generally as broad as the class ofhigher and lower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, and multicellular algae. It includes plants of a variety ofploidy levels, including aneuploid, polyploid, diploid, haploid andhemizygous.

The term “Bt toxin” refers to an insecticidal protein isolated orderived from Bacillus thuringiensis (Bt) bacteria. The term includesnaturally and non-naturally occurring variants, including fragments andmodified versions of naturally-occurring Bt toxins. (See, e.g., U.S.Pat. Nos. 6,489,542; 5,281,530; 5,322,932; U.S. patent application Ser.No. 11/067,557 filed Feb. 25, 2005; and PCT publication WO 92/04453.)

The term “recombinant” refers to a human-manipulated polynucleotide, acopy, or complement thereof. For instance, a recombinant expressioncassette comprising a promoter operably linked to a secondpolynucleotide may include a promoter that may be heterologous to thesecond polynucleotide as the result of human manipulation (e.g., bymethods described in Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)or Current Protocols in Molecular Biology Volumes 1-3, John Wiley &Sons, Inc. (1994-1998)). In another example, a recombinant expressioncassette may comprise polynucleotides combined in such a way that thepolynucleotides are extremely unlikely to be found in nature. Forinstance, human manipulated restriction sites or plasmid vectorsequences may flank or separate the promoter from the secondpolynucleotide. One of skill will recognize that polynucleotides can bemanipulated in many ways and are not limited to the examples above.

The term “variant polypeptide” refers to a polypeptide that is relatedto any one of SEQ ID NO:1-57 but has been altered in some respect (e.g.,deletion/addition of one or more residues, or making a derivative oranalog polypeptide). In some embodiments variant polypeptides have atleast partial plastid transit functional activity (e.g., the ability todirect an attached moiety into a plastid) of at least 50%, 60%, 70%,75%, 85%, 90%, 95%, 97%, 98%, or 99% when compared to the unalteredpolypeptide. In other embodiments, variant polypeptides have the same orbetter plastid transit functional activity when compared to theunaltered polypeptide. Generally, variant polypeptides are created inorder to accentuate a desirable characteristic (e.g., increase targetingefficiency, impart plastid specificity, make transcription and/ortranslation more efficient) or reduce an undesirable characteristic(e.g., degradation susceptibility) of a plastid transit peptide or apolypeptide comprising a plastid transit peptide. Variant polypeptidesdo not encompass any naturally occurring plastid transit peptides.

A variety of diversity generating protocols are available and describedin the art. See, e.g., Ling et al. (1997) Anal Biochem. 254(2): 157-178;Dale et al. (1996) Methods Mol. Biol. 57:369-374; U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, WO 95/22625, WO96/33207, WO 97/20078, WO 97/35966, WO 99/41402, WO 99/41383, WO99/41369, EP 752008, EP 0932670, WO 99/23107, WO 99/21979, WO 98/31837,WO 98/27230, WO 98/13487, WO 00/09679, WO 98/42832, WO 99/29902, WO98/41653, WO 98/41622, WO 00/42561, WO 00/42560, WO 01/75767 and WO98/42727.

The term “derivative polypeptide” refers to a polypeptide that isrelated to any one of SEQ ID NOS:1-57 but has been altered by one ormore amino acid residue changes yet retains at least partial plastidtransit functional activity. In some embodiments, the amino acid residuesubstituted is a chemically similar amino acid. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art (see, e.g., Creighton, Proteins (1984)). For example,the following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). In other embodiments, the amino acid residue substitutedis not a conservative substitution. Derivative polypeptides may haveless than 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1% of their residues alteredwhen compared to the unaltered polypeptide.

Sequence alterations can be introduced by standard techniques such asdirected molecular evolution techniques e.g., DNA shuffling methods (seee.g., Christians et al., 1999, Nature Biotechnology 17:259-264; Crameriet al., 1998, Nature, 391:288-291; Crameri, et al., 1997, NatureBiotechnology 15:436-438; Crameri et al., 1996, Nature Biotechnology14:315-319; Stemmer, 1994, Nature 370:389-391; Stemmer et al., 1994,Proc. Natl. Acad. Sci., 91:10747-10751; U.S. Pat. Nos. 5,605,793;6,117,679; 6,132,970; 5,939,250; 5,965,408; 6,171,820; InternationalPublication Nos. WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; and WO 01/75767); site-directed mutagenesis (see e.g., Kunkel,1985, Proc. Natl. Acad. Sci., 82:488-492; Oliphant et al., 1986, Gene44:177-183); oligonucleotide-directed mutagenesis (see e.g.,Reidhaar-Olson et al., 1988, Science 241:53-57); chemical mutagenesis(see e.g., Eckert et al., 1987, Mutat. Res. 178:1-10); error-prone PCR(see e.g., Caldwell & Joyce, 1992, PCR Methods Applic. 2:28-33); andcassette mutagenesis (see e.g., Arkin et al., Proc. Natl. Acad. Sci.,1992, 89:7871-7815); (see generally, e.g., Arnold, 1993, Curr. OpinionBiotechnol. 4:450-455; Ling et al., 1997, Anal. Biochem., 254(2):157-78;Dale et al., 1996, Methods Mol. Biol. 57:369-74; Smith, 1985, Ann. Rev.Genet. 19:423-462; Botstein et al., 1985, Science, 229:1193-1201;Carter, 1986, Biochem. J. 237:1-7; Kramer et al., 1984, Cell 38:879-887;Wells et al., 1985, Gene 34:315-323; Minshull et al., 1999, CurrentOpinion in Chemical Biology 3:284-290).

Additionally, the nucleic acid molecules that encode derivativepolypeptides can be codon-optimized, either wholly or in part. Becauseany one amino acid (except for methionine) is encoded by a number ofcodons, the sequence of the nucleic acid molecule may be changed withoutchanging the encoded amino acid. Codon optimization is when one or morecodons are altered at the nucleic acid level to coincide with or betterapproximate the codon usage of a particular host. The frequency ofpreferred codon usage exhibited by a host cell can be calculated byaveraging frequency of preferred codon usage in a large number of genesexpressed by the host cell. This analysis may be limited to genes thatare highly expressed by the host cell. U.S. Pat. No. 5,824,864, forexample, provides the frequency of codon usage by highly expressed genesexhibited by dicotyledonous plants and monocotyledonous plants. Thosehaving ordinary skill in the art will recognize that tables and otherreferences providing preference information for a wide range oforganisms are available in the art.

The term “analog polypeptide” refers to polypeptides that possessresidues that have been modified, i.e., by the covalent attachment ofany type of molecule. For example, but not by way of limitation, ananalog polypeptide may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. An analog polypeptide may be modified bychemical modifications using techniques known to those of skill in theart, including, but not limited to specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Furthermore, an analog of a polypeptide may contain one or morenon-classical amino acids.

The term “identical” in connection to nucleic acid molecules andpolypeptides refers to two sequences that have identical residues whenaligned for maximum correspondence as described below.

The term “percent identity” in connection to nucleic acid molecules andpolypeptides refers to the percent of residues in two sequences that areidentical when compared and aligned for maximum correspondence over acomparison window, as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.

When percentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

When percentage of sequence identity is used in reference to nucleicacid molecules, any method known in the art can be used. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection.

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments, or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “stringent conditions” in connection to nucleic acidhybridization refers to hybridization conditions under which a nucleicacid molecule will hybridize to its target nucleic acid molecule,typically in a complex mixture of nucleic acid molecules, but toessentially no other nucleic acids. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer nucleic acids hybridize specifically at higher temperatures.Extensive guides to the hybridization of nucleic acids can be found inthe art (e.g., Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993)).Generally, highly stringent conditions are selected to be about 5-10° C.lower than the thermal melting point (T_(m)) for the specific nucleicacid at a defined ionic strength pH. Low stringency conditions aregenerally selected to be about 15-30° C. below the T_(m). The T_(m) isthe temperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target nucleic acid at equilibrium (as the targetnucleic acids are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Hybridization conditions are typically thosein which the salt concentration is less than about 1.0 M sodium ion,typically about 0.01 to 1.0 M sodium ion concentration (or other salts)at pH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, and preferably 10 times backgroundhybridization. In one embodiment, stringent conditions include at leastone wash (usually 2) in 0.2×SSC at a temperature of at least about 50°C., usually about 55° C., or sometimes 60° C. or 65° C., for 20 minutes,or substantially equivalent conditions. In a specific embodiment, anucleic acid molecule of the invention specifically hybridizes followingat least one wash in 0.2×SSC at 55° C. for 20 minutes to a nucleic acidmolecule encoding any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57. In another embodiment,stringent conditions include hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC,0.1% SDS at 50-65° C.

The phrase “specifically hybridizes” refers to the binding, duplexing,or hybridizing of a molecule only to a particular nucleotide sequenceunder stringent hybridization conditions when that sequence is presentin a complex mixture (e.g., total cellular or library DNA or RNA).

The term “substantially similar” when used in connection with plastidtransit peptide functional activity refers to two plastid transitpeptides having a level of activity that is similar to each other. Insome embodiments, plastid transit peptides have substantially similaractivity when their activities, as measured in an assay, are onestandard deviation or less away from each other. In other embodiments,plastid transit peptides have substantially similar activity when one ofthe peptide's activity is at least 75%, 80%, 85%, 90%, 95%, 99% of theactivity of the other peptide as measured in the same assay.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel plastid transit peptides. Nucleicacid molecules encoding the polypeptides of the invention are alsoprovided. Methods for using the peptides and nucleic acid molecules ofthe invention to target polypeptides to plant plastids (e.g.,chloroplasts, etioplasts, amyloplasts, chromoplasts, elaioplasts, andleucoplasts) are encompassed.

4.1 Polypeptides of the Invention

The present invention relates to a novel plastid transit peptidesselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57. In addition to thepolypeptide sequences of SEQ ID NOS:1-57, it will be appreciated thatpeptides of the invention also encompass variants thereof, including,but not limited to, any fragment, derivative, or analog thereof. Inpreferred embodiments, the variant plastid transit peptides havesubstantially similar or improved activity when compared to non-variantplastid transit peptides.

In one embodiment, peptides encompassed by the present invention haveplastid transit functional activity (e.g., the ability to direct anattached moiety into a plastid) and are at least 85%, 90%, 95%, 97%,98%, or 99% identical to the peptide sequence of any of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQID NO:57.

In another embodiment, peptides encompassed by the present inventionhave plastid transit functional activity (e.g., the ability to direct anattached moiety into a plastid) and are a fragment comprising at least70%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% of the contiguous amino acidresidues of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, and SEQ ID NO:57.

In another embodiment, peptides encompassed by the present inventionhave plastid transit functional activity (e.g., the ability to direct anattached moiety into a plastid) and are encoded by a nucleic acidmolecule comprising a nucleotide sequence that is at least 95% identicalto any of the nucleic acid molecules that encode any of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQID NO:57.

Methods of production of the peptides of the invention and/orpolypeptides that comprise peptides of the invention, e.g., byrecombinant means, are provided (see Section 4.6).

Compositions comprising one or more peptides of the invention and/orpolypeptides that comprise peptides of the invention are alsoencompassed. The compositions of the invention can further compriseadditional agents including, but not limited to, spreader-stickeradjuvants, stabilizing agents, diluents, agents that optimize therheological properties or stability of the composition, such as, forexample, surfactants, emulsifiers, dispersants, and/or polymers.

4.2 Fusion Polypeptides

The present invention provides methods for targeting a polypeptide to aplant plastid by attaching a plastid transit peptide of the invention tothe polypeptide to be targeted. In preferred embodiments, the methodcomprises recombinantly attaching a first nucleic acid molecule encodinga plastid transit peptide of the invention to a second nucleic acidmolecule encoding a polypeptide to be targeted such that translation ofthe nucleic acid molecule produces a fusion polypeptide. The fusionpolypeptides are also encompassed by the present invention.

The plastid transit peptide is generally fused N-terminal to thepolypeptide to be targeted (e.g., the fusion partner). In oneembodiment, the fusion protein consists essentially of the peptidetransit plastid and the polypeptide to be targeted. In anotherembodiment, the fusion protein comprises the peptide transit plastid andthe polypeptide to be targeted. In such embodiments, the plastid transitpeptide is preferably at the N-terminus of the fusion protein. However,additional amino acid residues may be N-terminal to the plastid transitpeptide providing that the fusion protein is at least partially targetedto a plastid. In a specific embodiment, the plastid transit peptide isin the N-terminal half, N-terminal third, or N-terminal quarter of thefusion protein.

Most or all of the plastid transit peptide is generally cleaved from thefusion protein upon insertion into the plastid. The position of cleavagemay vary slightly between plant species, at different plantdevelopmental stages, as a result of specific intercellular conditions,or the particular combination of transit peptide/fusion partner used. Inone embodiment, the plastid transit peptide cleavage is homogenous suchthat the cleavage site is identical in a population of fusion proteins.In another embodiment, the plastid transit peptide is not homogenous,such that the cleavage site varies by 1-10 amino acids in a populationof fusion proteins.

The plastid transit peptide can be recombinantly fused to a secondprotein in one of several ways. For example, a restriction endonucleaserecognition site can be introduced into the nucleotide sequence of thetransit peptide at a position corresponding to its C-terminal end, andthe same or a compatible site can be engineered into the nucleotidesequence of the protein to be targeted at its N-terminal end. Care mustbe taken in designing these sites to ensure that the coding sequences ofthe transit peptide and the second protein are kept “in frame” to allowthe synthesis of the desired fusion protein. In some cases, it may bepreferable to remove the initiator methionine codon of the secondprotein when the new restriction site is introduced. The introduction ofrestriction endonuclease recognition sites on both parent molecules andtheir subsequent joining through recombinant DNA techniques may resultin the addition of one or more extra amino acids between the transitpeptide and the second protein. This generally does not affect targetingactivity as long as the transit peptide cleavage site remains accessibleand the function of the second protein is not altered by the addition ofthese extra amino acids at its N-terminus. Alternatively, one skilled inthe art can create a precise fusion between the transit peptide and thesecond protein (with or without its initiator methionine) using genesynthesis (Stemmer et al., Gene 164:49-53 (1995)) or similar methods.

In addition, the transit peptide fusion can intentionally include aminoacids downstream of the cleavage site. The amino acids at the N-terminusof the mature protein can affect the ability of the transit peptide totarget proteins to plastids and/or the efficiency of cleavage followingprotein import. This may be dependent on the protein to be targeted.See, e.g., Comai et al., J. Biol. Chem. 263(29):15104-9 (1988).

The fusion partner (e.g., the polypeptide to be targeted) may be anypolypeptide for which plastid localization is desired. Fusion partnersmay be full-length proteins (e.g., as they occur in nature) or may bemodified versions of such proteins (e.g., portions or fragments thereof,variants, or other non-naturally occurring versions of a protein).Fusion partners can be from any organism, including, but are not limitedto, proteins from bacteria, algae, yeast, plants, animals, as well assynthetic proteins. For example, polypeptides that may be included infusion proteins include, but are not limited to, Bt toxin proteins (see,e.g., U.S. Pat. Nos. 6,489,542; 5,281,530; 5,322,932; U.S. patentapplication Ser. No. 11/067,557 filed Feb. 25, 2005; and PCT publicationWO 92/04453); 5-enolpyruvyl-3-phosphoshikimate synthase (EPSP synthase)(see. e.g., U.S. Pat. Nos. 4,971,908; 6,225,114); glyphosate N-acetyltransferase (GAT) (see, e.g., U.S. Patent Publication No. 2003/0083480),acetolactate synthase (ALS) (see, e.g., U.S. Pat. No. 5,013,659),enzymes that modify a physiological process that occurs in a plastid(e.g., photosynthesis or fatty acid, amino acid, oil, carotenoid,terpenoid, starch composition/synthesis) including, but not limited to,rubisco, rubisco activase, fatty acid synthase, fatty acid desaturase,phytoene synthase, phytoene desaturase, starch synthase, ADP-glucosepyrophosphorylase.

Different plastid transit peptides have differing degrees of efficacy(e.g., higher ratio of targeted to non-targeted fusion partner) whenused in combination with different fusion partners. The particularplastid transit peptide to use in combination with a particular fusionpartner can be determined empirically using, e.g., the assays describedin section 4.4.

4.3 Nucleic Acid Molecules of the Invention

The present invention also relates to nucleic acid molecules that encodeany one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, and SEQ ID NO:57, or any variants (e.g., any fragment,derivative, or analog) thereof.

In one embodiment, nucleic acid molecules encompassed by the presentinvention have plastid transit functional activity (e.g., the ability todirect an attached moiety into a plastid) and hybridize under stringentconditions to any one of the nucleic acid molecules that encode any ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,and SEQ ID NO:57.

In another embodiment, nucleic acid molecules encompassed by the presentinvention have plastid transit functional activity (e.g., the ability todirect an attached moiety into a plastid) and are a fragment comprisingat least 70%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% of the contiguousnucleic acid residues of any one of the nucleic acid molecules thatencode any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, and SEQ ID NO:57.

In another embodiment, nucleic acid molecules encompassed by the presentinvention have plastid transit functional activity (e.g., the ability todirect an attached moiety into a plastid) and comprise a nucleotidesequence that encodes a peptide that is at least 85%, 90%, 95%, 97%,98%, or 99% identical to the amino acid sequence of any of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQID NO:57.

In another embodiment, nucleic acid molecules encompassed by the presentinvention have plastid transit functional activity (e.g., the ability todirect an attached moiety into a plastid) and comprise a nucleotidesequence which is at least 85%, 90%, 95%, 97%, 98%, or 99% identical toany of the nucleic acid molecules that encodes any of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQID NO:57.

Vectors or expression cassettes comprising nucleic acid molecules of theinvention are also encompassed (see Section 4.6). Cells, plants, orseeds comprising the vectors of the invention are also encompassed (seeSection 4.7).

4.4 Methods to Assay for Plastid Transit Peptide Activity

Plastid transit peptide function or activity can be assayed by anymethod known in the art (see e.g., Lee et al., 2002, Mol. Cells.14:388-97; Archer and Keegstra, 1993, Plant Mol. Biol. 23:1105-15; Reisset al., 1989, Proc Natl Acad Sci USA. 86:886-90, Rensink et al., 1998,Plant Physiol. 118:691-9; Kindle and Lawrence, 1998, Plant Physiol.116:1179-90; Jin et al., 2003, Plant Mol Biol. 51:493-507). As usedherein, plastid transit peptide activity or function refers to theability of a plastid transit peptide to direct an attached moiety (e.g.,polypeptide) to a plastid. When attached to a functioning plastidtransit peptide, the attached moiety is enriched (e.g., by at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a moietynot attached to the plastid transit peptide) in one or more plastids.

Typically, activity of a plastid transit peptide is compared to apositive (i.e., a transit peptide known to target the particular fusionpartner) and/or a negative control (i.e., the polypeptide lacking aplastid transit peptide or comprising a non-functional plastid transitpeptide). Assays for transit peptide activity may involve, but are notlimited to, constructing recombinant fusions between a candidate plastidtransit peptide and a fusion partner polypeptide and expressing thefusion in a plant or plant cell.

In one embodiment, the fusion polypeptide is functional only orsubstantially only in the plastid; thus, plastid localization of thefusion partner is determined by the functionality of the fusion partner.In a specific embodiment, the enzymatic activity (e.g., by making acalorimetric or other readily-detectable product) of the fusion partneris assayed. For example, lysine decarboxylase can be targeted toplastids and the accumulation of cadaverine monitored as an indicationof enzyme targeting efficiency (see, e.g., Herminghaus et al., 1991,Plant Mol. Biol. 17:475-486 and Herminghaus et al., 1996, TransgenicResearch 5:193-201). The conversion of L-trytophan to tryptamine byplastid-targeted trytophan decarboxylase can be measured as anindication of enzyme targeting efficiency (see, e.g., Fiore et al. 2002,Plant Physiol. 129:1160-1169). Changes in the distribution of existingcarotenoid pigments, or the accumulation of non-native carotenoids canbe examined as an indication of proper targeting and activity of variouscarotenoid biosynthetic enzymes (see, e.g., Kumagai et al., 1998, PlantJ 14:305-315).

In another embodiment, the fusion polypeptide is fluorescent; thus,plastid localization of the fusion partner is determined by monitoringthe accumulation of fluorescence in the plastids using, e.g., afluorescence microscope. A preferred fluorescent protein is greenfluorescent protein and variants thereof (see, e.g., Nakrieko et al.,2004, Eur J Biochem. 271:509-516; Belluci et al., 2003, Plant Cell Rep.22:328-337; Chiu et al., 1996, Curr Biol. 6:325-330).

In another embodiment, plastid localization of the fusion partner isdetermined by determining the size of the fusion partner. Plastidtransit peptides are typically cleaved in their entirety or in part whenthe fusion is inserted into a plastid. If the plastid transit peptidecontains a cleavage site that is accessible as part of the fusionprotein, then the plastid transit peptide will be cleaved off and thelength (and therefore the molecular weight) of the polypeptide will bedecreased. If the sequence of the cleavage site is not readilyaccessible, (e.g., if the surrounding sequences prevent properrecognition of the cleavage site or if the fusion protein folds in a waysuch that the stromal protease cannot gain access to the cleavage site)then cleavage will be inefficient and may occur at one or morealternative positions. Although the processed fusion partnerpolypeptides in this case will be of slightly varied length, they willstill all be decreased in length and molecular weight from unprocessedpolypeptide.

In another embodiment, plastids are isolated from plant tissue and thenassayed for the presence of the fusion partner polypeptide. Any methodknown in the art for polypeptide detection can be used to assay for thepresence of the fusion partner including, immunoblot,immunoprecipitation, ELISA, or detection of a trait of the fusionpartner (e.g., fluorescence or enzymatic activity).

A transit peptide is deemed to be functional if the level of end-productproduction, fluorescent protein accumulation inside plastids, or matureprotein accumulation in the above assays exceeds that of the negativecontrol. A plastid transit peptide is considered to be efficient if theabove parameters reach or exceed 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,99% of the level achieved by the positive control. In one embodiment,the plastid transit peptide used as a positive control is the transitpeptide from the ribulose bisphosphate carboxylase-oxygenase smallsubunit gene (see for example, Comai et al., J. Biol. Chem.263:15104-15109, 1988; Herminghaus et al., Plant Mol. Biol. 17:475-486,1991).

4.5 Methods of Use

Plastid transit peptides can be used to target an attached moiety (e.g.,polypeptide) to a plant plastid. In one embodiment, the plastid transitpeptide directs localization to all plastids in all tissue types. Inanother embodiment, the plastid transit peptide directs localization toa subset of plastids in all tissue types. In another embodiment, theplastid transit peptide directs localization to all plastids in a subsetof tissue types. In another embodiment, the plastid transit peptidedirects localization to a subset of plastids in a subset of tissuetypes.

In one embodiment, the attached polypeptide targeted to a plastid isinvolved in a physiological process that takes place in the plastid(including, but not limited to, photosynthesis or fatty acid, aminoacid, oil, carotenoid, terpenoid, starch composition/biosynthesis). Assuch, the targeted recombinant polypeptides can modulate or alter thephysiological processes that occur in the plastids (e.g., by alteringthe levels of the enzyme and/or providing an altered enzyme with afunction slightly different than the wild type enzyme). In a specificembodiment, the fusion partner is altered in a such a way to make theplant resistant to one or more herbicides. In a more specificembodiment, the fusion partner is acetolactate synthase (ALS) mutated tobe resistant to one or more herbicides (see, e.g., U.S. Pat. No.5,013,659).

In another embodiment, recombinant polypeptides are expressed anddirected to plastids by the methods of the present invention tofacilitate higher expression levels. For example, some polypeptides aretoxic when expressed recombinantly in the cytoplasm of a plant celland/or some polypeptides are sensitive to proteases and otherconstituents of the cytoplasm that case degradation. Because plastidsare subcellular compartments, it is possible to target recombinantpolypeptides to the plastids to sequester them from the cytoplasm, thusallowing for higher expression levels. In a specific embodiment, thefusion partner has insecticidal activity. In a more specific embodiment,the fusion partner is one or more Bt toxin proteins (see, e.g., U.S.Pat. Nos. 6,489,542; 5,281,530; 5,322,932; U.S. patent application Ser.No. 11/067,557 filed Feb. 25, 2005; and PCT publication WO 92/04453).

In another embodiment, recombinant polypeptides are expressed anddirected to plastids by the methods of the present invention to avoidadverse agronomic effects to the plant. Some polypeptides are toxic orcause undesirable plant phenotypes when expressed recombinantly in thecytoplasm of a plant cell. By sequestering the recombinant polypeptidesto the plastids, these unwanted effects can often be reduced oreliminated.

In another embodiment, recombinant polypeptides are expressed anddirected to plastids by the methods of the present invention tofacilitate easier isolation of the polypeptide. Plastids can be isolatedfrom plant tissue by any method known in the art and the polypeptidescontained in them extracted.

In another embodiment, recombinant polypeptides are expressed anddirected to plastids by the methods of the present invention tofacilitate higher expression levels. Some polypeptides are sensitive toproteases and other constituents of the cytoplasm. Because plastids aresubcellular compartments, it is possible to target recombinantpolypeptides to the plastids to sequester them from the cytoplasm, thusallowing for higher expression levels.

In another embodiment, recombinant polypeptides are expressed anddirected to plastids by the methods of the present invention to regulatetheir activity on a substrate(s) that is localized in a differentsubcellular compartment. By separating the polypeptides/enzymes andsubstrate(s) into different subcellular compartments, the activity ofthe enzymes can be controlled by processes such as heating, grinding, ormechanical extraction that result in mixing of the enzymes andsubstrate(s) together.

In another embodiment, a recombinant polypeptide that forms aheteromeric complex is expressed and directed to plastids by the methodsof the present invention to regulate the activity of the assembledenzyme complex. By separating components of the complex into differentsubcellular compartments, the activity of the assembled complex can becontrolled by processes such as heating, grinding, or mechanicalextraction that result in mixing of the polypeptide components together.

4.6 Recombinant Expression

Nucleic acid molecules and polypeptides of the invention can beexpressed recombinantly using standard recombinant DNA and molecularcloning techniques that are well known in the art (e.g., Sambrook,Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, 1989). Additionally,recombinant DNA techniques may be used to create nucleic acid constructssuitable for use in making transgenic plants (see Section 4.7).

Accordingly, an aspect of the invention pertains to vectors, preferablyexpression vectors, comprising a nucleic acid molecule of the invention,or a variant thereof. As used herein, the term “vector” refers to apolynucleotide capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe introduced. Another type of vector is a viral vector, whereinadditional DNA segments can be introduced into the viral genome.

Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal vectors). Other vectors(e.g., non-episomal vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses).

The recombinant expression vectors of the invention comprise a nucleicacid molecule of the invention in a form suitable for expression of thenucleic acid molecule in a host cell. This means that the recombinantexpression vectors include one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which is operablyassociated with the polynucleotide to be expressed. Within a recombinantexpression vector, “operably associated” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described in the art (e.g., Goeddel, GeneExpression Technology: Methods in Enzymology, 1990, Academic Press, SanDiego, Calif.). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, thearea of the organism in which expression is desired, etc. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids molecules as described herein.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, U.S. Pat. No. 5,565,350; International PatentApplication No. PCT/US93/03868), or isolated promoters can be introducedinto a plant cell in the proper orientation and distance from a cognategene of a polynucleotide of the present invention so as to control theexpression of the gene. Gene expression can be modulated underconditions suitable for plant growth so as to alter the totalconcentration and/or alter the composition of the polypeptides of thepresent invention in plant cell. Thus, the present invention providescompositions, and methods for making heterologous promoters and/orenhancers operably linked to a native, endogenous (i.e.,non-heterologous) form of a polynucleotide of the present invention.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide of the invention in prokaryotic (e.g.,Enterobacteriaceae, such as Escherichia; Bacillaceae; Rhizoboceae, suchas Rhizobium and Rhizobacter; Spirillaceae, such as photobacterium;Zymomonas; Serratia; Aeromonas; Vibrio; Desulfovibrio; Spirillum;Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter;Azotobacteraceae and Nitrobacteraceae) or eukaryotic cells (e.g., insectcells using baculovirus expression vectors, yeast cells, plant cells, ormammalian cells) (see Goeddel, supra. For a discussion on suitable hostcells). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors comprising constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve at least three purposes: 1) to increaseexpression of the recombinant protein; 2) to increase the solubility ofthe recombinant protein; and/or 3) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase. Typical fusion expression vectors include pGEX (PharmaciaBiotech Inc; Smith and Johnson, 1988, Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corp., San Diego, Calif.), and pPicZ(Invitrogen Corp., San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin plant cells using a plant expression vector including, but notlimited to, tobacco mosaic virus and potato virus expression vectors.

Other suitable expression systems for both prokaryotic and eukaryoticcells are known in the art (see, e.g., chapters 16 and 17 of Sambrook etal. 1990, Molecular Cloning A Laboratory Manual, 2d Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-specific, inducible, orother promoters for expression in the host organism.

A “tissue-specific promoter” may direct expression of nucleic acids ofthe present invention in a specific tissue, organ or cell type.Tissue-specific promoters can be inducible. Similarly, tissue-specificpromoters may only promote transcription within a certain time frame ordevelopmental stage within that tissue. Other tissue specific promotersmay be active throughout the life cycle of a particular tissue. One ofordinary skill in the art will recognize that a tissue-specific promotermay drive expression of operably linked sequences in tissues other thanthe target tissue. Thus, as used herein, a tissue-specific promoter isone that drives expression preferentially in the target tissue or celltype, but may also lead to some expression in other tissues as well. Anumber of tissue-specific promoters can be used in the presentinvention. With the appropriate promoter, any organ can be targeted,such as shoot vegetative organs/structures (e.g. leaves, stems andtubers), roots, flowers and floral organs/structures (e.g. bracts,sepals, petals, stamens, carpels, anthers and ovules), seed (includingembryo, endosperm, and seed coat) and fruit. For expression of apolynucleotide of the present invention in the aerial vegetative organsof a plant, photosynthetic organ-specific promoters, such as the RBCSpromoter (Khoudi et al., Gene 197:343, 1997), can be used. Root-specificexpression of polynucleotides of the present invention can be achievedunder the control of a root-specific promoter, such as, for example, thepromoter from the ANR1 gene (Zhang and Forde, Science, 279:407, 1998).Other exemplary promoters include the root-specific glutamine synthetasegene from soybean (Hirel et al., 1992, Plant Molecular Biology20:207-218) and the root-specific control element in the GRP 1.8 gene ofFrench bean (Keller et al., 1991, The Plant Cell 3:1051-1061).

A “constitutive promoter” is defined as a promoter which will directexpression of a gene in all tissues and are active under mostenvironmental conditions and states of development or celldifferentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, andother transcription initiation regions from various plant genes known tothose of ordinary skill in the art. Such genes include for example,ACT11 from Arabidopsis (Huang et al. 1996, Plant Mol. Biol. 33:125-139),Cat3 from Arabidopsis (Genbank Accession No. U43147, Zhong et al., 1996,Mol. Gen. Genet. 251:196-203), the gene encoding stearoyl-acyl carrierprotein desaturase from Brassica napus (Genbank Accession No. X74782,Solocombe et al. 1994, Plant Physiol. 104:1167-1176), GPc1 from maize(GenBank Accession No. X15596, Martinez et al., 1989, J. Mol. Biol.208:551-565), and Gpc2 from maize (GenBank Accession No. U45855,Manjunath et al., 1997, Plant Mol. Biol. 33:97-112). Any strong,constitutive promoter, such as the CaMV 35S promoter, can be used forthe expression of polynucleotides of the present invention throughoutthe plant.

The term “inducible promoter” refers to a promoter that is under preciseenvironmental or developmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions, elevated temperature, the presence of light, orspraying with chemicals/hormones.

Suitable constitutive promoters for use in a plant host cell include,for example, cauliflower mosaic virus (CaMV) 35S transcriptioninitiation region, the full-length transcript promoter of mirabilismosaic virus (Dey and Maiti, Plant Mol. Biol. 40:771-782, (1999)), the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, thefull-length transcript promoter from peanut chlorotic streak virus(Maiti and Shepherd, Biochem. Biophys. Res. Comm. 244:440-444 (1998)),the 34S promoter from figwort mosaic virus (Maiti et al., Transgen. Res.6:143-156 (1997); Sanger et al., Plant Mol. Biol. 14:433-443 (1990)),and the full-length transcript promoter from strawberry vein bandingvirus (U.S. Patent Publication No. 2002/0182593) as well as othertranscription initiation regions from various plant genes known to thoseof skill. Such genes include for example, ACT11 from Arabidopsis (Huanget al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis(GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)),the gene encoding stearoyl-acyl carrier protein desaturase from Brassicanapus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176(1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol.Biol. 208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855,Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)), rice actin(McElroy et al., 1990, Plant Cell 2:163-171); ubiquitin (Christensen etal., 1989, Plant Mol. Biol. 12:619-632 and Christensen et al., 1992,Plant Mol. Biol. 18:675-689); pEMU (Last et al., 1991, Theor. Appl.Genet. 81:581-588).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

Accordingly, the present invention provides a host cell having anexpression vector comprising a nucleic acid of the invention, or avariant thereof. A host cell can be any prokaryotic (e.g., E. coli,Bacillus thuringiensis) or eukaryotic cell (e.g., insect cells, yeast orplant cells). The invention also provides a method for expressing anucleic acid of the invention thus making the encoded polypeptidecomprising the steps of i) culturing a cell comprising a nucleic acidmolecule of the invention under conditions that allow production of theencoded polypeptide; and ii) isolating the expressed polypeptide.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid molecules into a host cell, including calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in the art (e.g., Sambrook, et al.supra.).

4.7 Production of Transgenic Plants

Any method known in the art can be used for transforming a plant orplant cell with a nucleic acid molecule of the present invention.Nucleic acid molecules can be incorporated into plant DNA (e.g., genomicDNA or chloroplast DNA) or be maintained without insertion into theplant DNA (e.g., through the use of artificial chromosomes). Suitablemethods of introducing nucleic acid molecules into plant cells includemicroinjection (Crossway et al., 1986, Biotechniques 4:320-334);electroporation (Riggs et al., 1986, Proc. Natl. Acad. Sci.83:5602-5606; D'Halluin et al., 1992, Plant Cell 4:1495-1505);Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840, Osjoda et al., 1996, Nature Biotechnology 14:745-750; Horschet al., 1984, Science 233:496-498, Fraley et al., 1983, Proc. Natl.Acad. Sci. 80:4803, and Gene Transfer to Plants, Potrykus, ed.,Springer-Verlag, Berlin 1995); direct gene transfer (Paszkowski et al.,1984, EMBO J. 3:2717-2722); ballistic particle acceleration (U.S. Pat.Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., 1995,“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment, in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips, Springer-Verlag, Berlin; and McCabeet al., 1988, Biotechnology 6:923-926); virus-mediated transformation(U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and5,316,931); pollen transformation (De Wet et al., 1985, in TheExperimental Manipulation of Ovule Tissues, ed. Chapman et al., Longman,N.Y., pp. 197-209); Lec 1 transformation (U.S. patent application Ser.No. 09/435,054; International Publication No. WO 00/28058);whisker-mediated transformation (Kaeppler et al., 1990, Plant CellReports 9:415-418; Kaeppler et al., 1992, Theor. Appl. Genet.84:560-566); and chloroplast transformation technology (Bogorad, 2000,Trends in Biotechnology 18: 257-263; Ramesh et al., 2004, Methods MolBiol. 274:301-7; Hou et al., 2003, Transgenic Res. 12:111-4; Kindle etal., 1991, Proc. Natl. Acad. Sci. 88:1721-5; Bateman and Purton, 2000,Mol Gen Genet. 263:404-10; Sidorov et al., 1999, Plant J. 19:209-216).

The choice of transformation protocols used for generating transgenicplants and plant cells can vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Examples oftransformation protocols particularly suited for a particular plant typeinclude those for: potato (Tu et al., 1998, Plant Molecular Biology37:829-838; Chong et al., 2000, Transgenic Research 9:71-78); soybean(Christou et al., 1988, Plant Physiol. 87:671-674; McCabe et al., 1988,BioTechnology 6:923-926; Finer and McMullen, 1991, In Vitro Cell Dev.Biol. 27P:175-182; Singh et al., 1998, Theor. Appl. Genet. 96:319-324);maize (Klein et al., 1988, Proc. Natl. Acad. Sci. 85:4305-4309; Klein etal., 1988, Biotechnology 6:559-563; Klein et al., 1988, Plant Physiol.91:440-444; Fromm et al., 1990, Biotechnology 8:833-839; Tomes et al.,1995, “Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin)); cereals (Hooykaas-VanSlogteren et al., 1984, Nature 311:763-764; U.S. Pat. No. 5,736,369).

In some embodiments, more than one construct is used for transformationin the generation of transgenic plants and plant cells. Multipleconstructs may be included in cis or trans positions. In preferredembodiments, each construct has a promoter and other regulatorysequences.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker that has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in the art (e.g., Evans et al., ProtoplastsIsolation and Culture, Handbook of Plant Cell Culture, pp. 124-176,MacMillilan Publishing Company, New York, 1983; and Binding,Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, BocaRaton, 1985). Regeneration can also be obtained from plant callus,explants, organs, or parts thereof. Such regeneration techniques arealso described in the art (e.g., Klee et al. 1987, Ann. Rev. of PlantPhys. 38:467-486).

The nucleic acid molecules of the invention can be used to target apolypeptide to a plastid in essentially any plant. Thus, the inventionhas use over a broad range of plants, including species from the generaAgrotis, Allium, Ananas, Anacardium, Apium, Arachis, Asparagus,Athamantha, Atropa, Avena, Bambusa, Beta, Brassica, Bromus, Browaalia,Camellia, Cannabis, Carica, Ceratonia. Cicer, Chenopodium, Chicorium,Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Coix, Cucumis,Cucurbita, Cynodon, Dactylis, Datura, Daucus, Dianthus, Digitalis,Dioscorea, Elaeis, Eliusine, Euphorbia, Festuca, Ficus, Fragaria,Geranium, Glycine, Graminae, Gossypium, Helianthus, Heterocallis, Hevea,Hibiscus, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lathyrus, Lens, Lilium,Linum, Lolium, Lotus, Lupinus, Lycopersicon, Macadamia, Macrophylla,Malus, Mangifera, Manihot, Majorana, Medicago, Musa, Narcissus, Nemesia,Nicotiana, Onobrychis, Olea, Olyreae, Oryza, Panicum, Panicum, Panieum,Pannisetum, Pennisetum, Petunia, Pelargonium, Persea, Pharoideae,Phaseolus, Phleum, Picea, Poa, Pinus, Pistachia, Pisum, Populus,Pseudotsuga, Pyrus, Prunus, Pseutotsuga, Psidium, Quercus, Ranunculus,Raphanus, Ribes, Ricinus, Rhododendron, Rosa, Saccharum, Salpiglossis,Secale, Senecio, Setaria, Sequoia, Sinapis, Solanum, Sorghum,Stenotaphrum, Theobromus, Trigonella, Trifolium, Trigonella, Triticum,Tsuga, Tulipa, Vicia, Vitis, Vigna, and Zea.

In specific embodiments, transgenic plants are maize, tomato, potato,rice, soybean, cotton plants, sunflower, alfalfa, lettuce, or tobacco.

Transgenic plants may be grown and pollinated with either the sametransformed strain or different strains. Two or more generations of theplants may be grown to ensure that expression of the desired nucleicacid molecule, polypeptide and/or phenotypic characteristic is stablymaintained and inherited. One of ordinary skill in the art willrecognize that after the nucleic acid molecule of the present inventionis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

4.8 Determination of Expression in Transgenic Plants

Any method known in the art can be used for determining the level ofexpression in a plant of a nucleic acid molecule of the invention orpolypeptide encoded therefrom. For example, the expression level in aplant of a polypeptide encoded by a nucleic acid molecule of theinvention can be determined by immunoassay, immunoblot, quantitative gelelectrophoresis, etc.

Additionally, the expression level in a plant of a polypeptide encodedby a nucleic acid molecule of the invention can be determined by thedegree to which the phenotype of the transgenic plant is altered. In aspecific embodiment, enhanced polypeptide targeting to plastids is thephenotype to be assayed. Such phenotypes include, but are not limitedto, a change in the amount or composition of fatty acids, amino acids,oils, terpenoids, or starch in seeds or other tissues, enhancedtolerance to an applied herbicide, greater resistance to a pest (e.g.,insect and/or nematode), and/or increase in harvestable seed/grain yieldand/or plant biomass.

Determinations can be made using whole plants, tissues thereof, plantcell culture, or plastids purified therefrom.

The contents of all published articles, books, reference manuals andabstracts cited herein, are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,and/or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Modifications and variations ofthe present invention are possible in light of the above teachings.

5. EXAMPLES

The following example is offered to illustrate, but not to limit theclaimed invention.

5.1 Example 1

A library of non-naturally occurring peptide sequences was recombinantlyfused to a lysine decarboxylase (LDC) gene from E. coli and transientlyexpressed in Nicotiana tabacum BY-2 suspension cells viaAgrobacterium-mediated transformation (see generally Newman et al.,Plant Cell 5:701-714, 1993). After 4 days the cells were rapidlyfreeze-thawed, re-hydrated in water+0.5% formic acid for at least onehour, dispersed with a 96-well replicator pin device, and then thesupernatants were collected by spinning the mixtures through a MilliporeMAHVN45 filter plate. Dilutions of the supernatants were analyzed forthe presence of the end-product cadaverine using LC-Mass Spectroscopy(MS) as follows:

A triple quadrupole MS instrument (Quattro LC, Micromass) equipped withelectrospray LC/MS interface was connected with an HPLC pump (Agilent1050) delivering 40/60 of H₂O/MeOH w/0.1% formic acid at 0.3 ml/minconstant flow rate. A flow injection method was used in analysis withTwin Pal auto injector (Leap Technology), injecting 5 μl of samplesolution into the MS with a rate of one-half minute per injection. TheMass Spectrometer was operated at MRM mode for quantification ofcadaverine (MS/MS transaction: 102.8>85.8) and D-lysine (MS/MStransaction: 146.8>83.6). Peak heights and areas were determined fortransit peptide library clones and compared to positive (tobacco smallsubunit transit peptide) and negative (no transit peptide) controls.(For additional background information, see Herminghaus et al., PlantMol. Biol. 17:475-486, 1991, and Herminghaus et al., Transgenic Research5:193-201, 1996.)

Using the above-described assay, the peptide sequences depicted in Table1 were found to efficiently target the lysine decarboxylase protein toplastids when fused to the N-terminus of this protein.

TABLE 1 Efficient plastid transit peptides identified with an LDC fusionpartner SEQ ID Clone NO Sequence sCTP-6H1 1MAATTLTSALPGAFSSSQRPSAPFNLQRSPRVLR RFNRKTGRQPRGLVRAAKAQ sCTP-8B6 2MAATAVTSASLGAFSSSQRPGASSNSQRSPRLLR RFNRKTGRQPRGLVLAAKAQ sCTP-1F7 3MAATAVSSVLPGAFSSSQRSSSPFNSQRSLIVLR RFNRKRRRQRRGRVLAAKAQ sCTP-1H1 4MAATTVSSALLSAFSSSQSPSASFSLQTLPIVLR RFNRKTGRKPRGRVLAAKAQ sCTP-2B4 5MAATTLTSASPSAFSSSQSSGAPSNLQRSLRLLR RFNRKTGRQRLGRIRAAKAQ sCTP-2C7 6MASSALSSASPGAFSSSQRPSAPFNLKTSPIVLR RFNRNTGRQPRRRIRAAKAQ sCTP-2E7 7MAASALSSASLSAFSSSQSSSAPSSSKTSLRVLR RFNRKRGRQPRGLIRAAKAQ sCTP-2F2 8MAATAVTSASLGAFSSSQSPSAPSSSKKSLRVLR RFNRKTGRKPRGRVRAAKAQ sCTP-2G8 9MASTAVSSASPGAFSSSQSSGAPSNLQRSPILLR RFNRKRGRKPLGRIRAAKAQ sCTP-2G9 10MASTTLTSASPSAFSSSQRPSAPSNSQRSPRVLR RFNRKRGRKPLRRVLAAKAQ sCTP-3B9 11MAATALTSVLPGAFSSSQSPSAPFSLQRSPIVLR RFNRNRGRQPRGRVRAAKAQ sCTP-3C1 12MAASALTSASLGAFSSSQRPSAPSNLQTSPIVLR RFNRKTGLQPRRRVRAAKAQ sCTP-3C12 13MAATALTSASPSAFSSSQRPGAPSSSKTSPRLLR RFNRNTRRQRRGLVRAAKAQ sCTP-3E10 14MASTAVSSASLGAFSSSQSSGASSSSKTLPILLR RFNRKTRRQPLRLVRAAKAQ sCTP-3E12 15MAASALTSASLGAFSSSQSPGAPSSSQTSLRVLR RFNRKTGPQRLRRVRAAKAQ sCTP-3E7 16MASTALSSASPGAFSSSQRPSSPSSSKTSLRVLR RFNRKTGLQRRGLVRAAKAQ sCTP-3E9 17MASSALSSASPGAFSSSQRPGSSSSSQTSPILLR RFNRKTGRQRLRRVRAAKAQ sCTP-3F7 18MAASALTSALPGAFSSSQRPSAPSSSQRLPRLLR RFNRNTGRQRLRRIRAAKAQ sCTP-4B7 19MASTAVTSVSPSAFSSSQRPGAPSSLQRSPRVLR RFNRKTGRQRLGLVLAAKAQ sCTP-4D6 20MASTAVSSALPSAFSSSQRSSSPSSLQTLPRLLR RFNRKRGRQRRRRVRAAKAQ sCTP-4E1 21MAASTVSSVSPSAFSSSQRPGAPFSSQRLPRVLR RFNRNTRRQRRGRVLAAKAQ sCTP-4E7 22MASTALTSALLGAFSSSQRPGASSSLKRSPRVLR RFNRNRRLKRLGRVRAAKAQ sCTP-4F1 23MASTTVSSASPGAFSSSQRSSSPSNSQTSPRVLR RFNRKTGRKPRGLVRAAKAQ sCTP-4F12 24MAATAVTSALPGAFSSSQRPSAPFNSKTSPIVLR RFNRKTGRQPRRRVRAAKAQ sCTP-5D1 25MAASTLSSVSPGAFSSSQSPGAPSSSQRSPRVLR RFNRNTGLQPRGRIRAAKAQ sCTP-5E1 26MASSALTSASPGAFSSSQRPSAPFNSQRSPILLR RFNRNTRRQRRGLIRAAKAQ sCTP-5G1 27MAASALTSVSLSAFSSSQRPGAPSSLKTSPRLLR RFNRNTGLQRRGRVRAAKAQ sCTP-5H1 28MASTAVSSALLSAFSSSQSSGSPFSSQTLLRLLR RFNRNTGRQPLRRVLAAKAQ sCTP-5H10 29MAATALTSASLGAFSSSQRSGSPSNSQTLPIVLR RFNRKTRLKPRGRVLAAKAQ sCTP-5H2 30MASSAVTSALPGAFSSSQSPSAPSSSKRLPIVLR RFNRKTGRKPRGLVRAAKAQ sCTP-5H5 31MAASALTSVSPGAFSSSQSPGAPSNSQTSLRVLR RFNRNTRRKPRGLVRAAKAQ sCTP-5H6 32MAATALTSASLGAFSSSQRPGSSSNSQTSPILLR RFNRKTRLQRRRRVRAAKAQ sCTP-6B1 33MAATTVTSASLGAFSSSQSPSAPFNSQTSPRVLR RFNRKTGRQPRGRVRAAKAQ sCTP-6F1 34MASSTLTSALPGAFSSSQSSSASSSSQTSLRVLR RFNRKTGLKRLGRVRAAKAQ sCTP-6G2 35MAASALTSASLSAFSSSQSSGASSSSQRSLRVLR RFNRKTGRQRRRRVLAAKAQ sCTP-7D6 36MASTTVSSASPGAFSSSQRPGASSSLQRSPRVLR RFNRNRGRQRRGRVLAAKAQ sCTP-7H1 37MASTTLSSASPGAFSSSQSPSAPFSSQRSLRVLR RFNRKRGRQPRGLVRAAKAQ sCTP-8H1 38MASTTLSSASLGAFSSSQSPSAPFSSQRLLRVLR RFNRKRGRKPRGRVRAAKAQ sCTP-5G11 39MASTTLSSASLASVSLGAFSSSQSPSAPSSSQTS PIVLRRFNRNTGRQPRRLVRAAKAQ

5.2 Example 2

A library of non-naturally occurring peptide sequences was recombinantlyfused to a Cry2 Bt toxin and transiently expressed in Nicotianabenthamiana leaves via Agrobacterium-mediated transformation (see Kapilaet al., Plant Science 122:101-108, 1997). Protein was extracted from theinfiltrated leaf tissue and analyzed by SDS-PAGE and western blotting.Since processing of proteins targeted to a plastid involves cleavage ofthe transit peptide sequence from the remainder of the protein, adecrease in molecular weight of the Cry2 protein relative to the initialtransit peptide-Cry2 fusion is an indication that the peptide sequencemediated proper targeting to plastids and subsequent cleavage uponimport. Using the above-described assay, the peptide sequences depictedin Table 2 were found to efficiently target the Cry2 protein to plastidswhen fused to the N-terminus of this protein.

TABLE 2 Efficient plastid transit peptides identified with a Cry2 fusionpartner SEQ ID Clone NO Sequence sCTP-6H1 1MAATTLTSALPGAFSSSQRPSAPFNLQRSPRVLR RFNRKTGRQPRGLVRAAKAQ sCTP-20 40MAASTLSSASPSAFSSSQRPSAPSSLKTSLIVLR RFNRKTGRQPRGLVLAAKAQ sCTP-A1 41MAASTLSSVSPGAFSSSQRSGAPSNLQRSPILLR RFNRKTGRQPRGRVRAAKAQ sCTP-28 42MAATTVSSALPGAFSSSQSSGSSFNSKTLPRVLR RFNRNTGRQPLGLVRAAKAQ sCTP-27 43MASTAVTSALPGAFSSSQSPSAPSSLQTSPILLR RFNRNRGLKRLGRIRAAKAQ sCTP-F1 44MASSALTSASPSAFSSSQSSSAPFNSQTSPIVLR RFNRNTGRQRRGRVLAAKAQ sCTP-G4 45MASSAVTSASPSAFSSSQSPSAPFNSKRSPILLR RFNRKTGLQPRRLVRAAKAQ sCTP-17 46MAATALTSALPGAFSSSQSPGAPSNLQTSPIVLR RFNRNTGRKPRGRILAAKAQ sCTP-5 47MAATTLSSALPGAFSSSQSSSAPSNSQTSPILLR RFNRKTGLQPRRRVLAAKAQ sCTP-11 48MAATALSSASLGAFSSSQRPGASSSLQRSLIVLR RFNRKTGRQRRGRVLAAKAQ sCTP-12 49MASSAVTSASLSAFSSSQRPSASFNLQTSPRVLR RFNRKTGRQRLGLVRAAKAQ sCTP-19 50MAATALTSALLGAFSSSQSPGASSSLQTSLIVLR RFNRNRGRQPRGRILAAKAQ sCTP-21 51MAASTLSSVSPGAFSSSQSPGAPSSSQRSPRVLR RFNRNTGLQPRGRIRAAKAQ sCTP-30 52MAASAVSPGAFSSSQSPGASSNSQRLLRVLR RFNRKTGLQPLGRIRAAKAQ sCTP-G1 53MAATALSSASPGAFSSSQRPSAPSNSQTLPRVLR RFNRNTRRQPRGLVLAAKAQ sCTP-A2 54MAATAVSSASPGAFSSSQRSSAPSSSQRLPIVLR RFNRKRGRQRRGLVLAAKAQ SCTP-G2 55MAASALTSVLPGAFSSSQRPSAPSNSKRLPRLLR RFNRNTGLQPRGRILAAKAQ sCTP-D3 56MASSALSSASLGAFSSSQSPSASFSSQTSPRLLR RFNRKTGLKRLGRVRAAKAQ

5.3 Example 3

A small subset of the synthetic plastid transit peptides listed in Table1 was tested for efficacy with a glyphosate acetyl transferase protein(see Science 304:1151-1154, 2004) using the Nicotiana benthamiana leafinfiltration assay described in Example 2. Using this assay, plastidtransit peptides sCTP-6H1 and sCTP-6F1 were found to target the GATprotein to plastids with reasonable efficiency when fused to theN-terminus of this protein (data not shown).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule comprising a nucleotide sequence thatencodes a peptide of SEQ ID NO: 43; b) a nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thenucleic acid molecule that encodes the peptide of SEQ ID NO: 43; c) anucleic acid molecule comprising a nucleotide sequence that encodes apeptide that is at least 95% identical to the amino acid sequence of SEQID NO: 43; and d) a nucleic acid molecule comprising a nucleotidesequence that hybridizes with a full-length complement of a nucleotidesequence that encodes the peptide of SEQ ID NO: 43, whereinhybridization conditions comprise two washes in 0.2× SSC at 65° C. for20 minutes each; and wherein each of the peptides encoded by the nucleicacid molecule of a), b), c) or d) targets a translationally fusedpolypeptide into plant plastids.
 2. An isolated nucleic acid moleculeencoding a fusion polypeptide comprising the nucleic acid molecule ofclaim
 1. 3. A vector comprising the nucleic acid molecule of claim
 2. 4.A plant cell which comprises the vector of claim
 3. 5. A transgenicplant comprising the nucleic acid molecule of claim
 2. 6. The transgenicplant of claim 5, wherein the plant is selected from the groupconsisting of maize, soybean, tomato, potato, cotton, sunflower,alfalfa, lettuce, tobacco, and rice.
 7. A method for targeting apolypeptide to a plastid in a plant comprising introducing into theplant a vector comprising a first nucleic acid molecule encoding aplastid peptide linked to a second nucleic acid molecule encoding saidpolypeptide such that translation of the first and second nucleic acidmolecule produces a fusion protein, wherein said first nucleic acidmolecule is the nucleic acid molecule of claim
 1. 8. The method of claim7 wherein the plastid transit peptide is N-terminal to the polypeptidein the fusion protein.
 9. The method of claim 7 wherein the polypeptideis selected from the group consisting of Bt toxin proteins, EPSPsynthase, GAT, ALS, and enzymes that modify a physiological process thatoccurs in a plastid.
 10. The method of claim 9 wherein the physiologicalprocess is photosynthesis, fatty acid synthesis, amino acid synthesis,oil synthesis, carotenoid synthesis, terpenoid synthesis, and starchsynthesis.
 11. The method of claim 7 wherein the polypeptide is isolatedfrom the plant plastids.